The mitochondrial network structure is affected by Sam50 depletion. (A) tom40kd-2 and sam50kd-2 cells were cultivated in the presence or absence of doxycycline (Dox) for 7 days. Mitochondria in cells untreated (−Dox) or treated with Dox (+Dox) were stained with 150 nM MitoTracker Orange and analyzed by confocal microscopy. DIC, differential interference contrast. Scale bar, 10 μm. Western blots show the level of knockdown of the respective protein. Tom40, translocase of the outer mitochondrial membrane 40 protein; Sam50, sorting and assembly machinery 50 protein; F1α, subunit α of F₁F_O ATPase. (B) One hundred cells from 3 independent experiments were quantified according to the appearance of their mitochondria. The graph shows the mean values ± SD.

Mitochondria lacking Sam50 show an altered structure of cristae. (A and B) sam50kd-2 and pLV-THM cells were induced with doxycycline for 7 days and fixed with glutaraldehyde. Noninduced (−Dox) and induced (+Dox) cells were embedded in resin, and ultrathin sections were analyzed by TEM. Scale bar, 1 μm. For quantification, at least 50 mitochondria were counted from several sections of two independent samples. The graphs show the mean values ± SD. (C) The size of 170 mitochondria from sam50kd-2 cells without (−Dox) or after 7 days of (+Dox) Dox treatment was determined from TEM pictures using the ImageJ software program. (D) Levels of the mitochondrial fusion proteins mitofusins 1 and 2 and an organizer of cristae, OPA1, were assessed by SDS-PAGE and Western blotting of mitochondria from sam50kd-2 cells with or without Dox induction for 7 days. F1α was used as a loading control. The arrow marks the specific mitofusin 1 band as indicated by the antibody manufacturer.

Reduction of Tom40, VDAC1, and metaxins does not affect the structure of cristae. (A, B, and C) tom40kd-2 cells were treated for 5 days and VDAC1kd-1 and mtx2kd-2 cells were treated for 7 days with doxycycline (Dox). Cells were then analyzed by TEM, and the effect on the morphology of cristae was quantified by counting at least 50 mitochondria from several sections of two different samples. Graphs represent mean values ± SD. Scale bar, 1 μm. The knockdown of the respective protein was assessed by SDS-PAGE and Western blotting. ICDH, isocitrate dehydrogenase; Hsp60, heat shock protein 60. The asterisk represents a cross-reactive band used as a loading control. The efficiency of the metaxin 2 knockdown was assessed by monitoring the reduction in the amount of metaxin 1.

Depletion of Sam50 affects levels of several other mitochondrial proteins. (A, B, and C) sam50kd-2 cells were grown in the absence (−Dox) or presence (+Dox) of doxycycline for 5, 7, and 28 days. Mitochondria were prepared from noninduced and induced samples, and 25 and 50 μg of mitochondrial protein was analyzed by SDS-PAGE and Western blotting. NDUFS1, Fe-S protein 1 of complex I; CoxII, subunit 2 of complex IV; CoreI, core protein 1 of complex III; SDHA, subunit A flavoprotein of complex II; Cyt c, cytochrome c. For other designations, see the text and the legends for and . (D) Mitochondria from panel A were analyzed by TEM, and the number of morphologically changed mitochondria was quantified by counting at least 50 mitochondria from different sections of two independent samples. The graph shows the mean values ± SD.

Depletion of mitofilin and metaxins affects mitochondrial proteins in a way similar to that with Sam50 reduction. (A and B) Mitochondria from mtx2kd-2 cells were isolated after doxycycline (Dox) treatment for 7 and 28 days. Protein levels of 25 and 50 μg of mitochondrial protein were analyzed by SDS-PAGE and Western blotting. (C and D) Knockdown of mitofilin was induced for 7 and 21 days with Dox, and mitochondria were isolated from noninduced and induced samples and analyzed by SDS-PAGE and Western blotting. For designations, see the text and the legends for , , and .

Sam50 and mitofilin are found together in a high-molecular-mass protein complex. (A) Mitochondria were isolated from mflkd-2, sam50kd-2, and mtx2kd-2 cells where the knockdown of the respective protein was induced with doxycycline (Dox) for 7 days. Fifty micrograms of mitochondrial protein was solubilized in 1% digitonin buffer and analyzed by BN-PAGE and Western blotting with antibodies against mitofilin and Tom40. An asterisk marks the larger mitofilin complex that is sometimes observed. (B) Fifty micrograms of mitochondria isolated from sam50kd-2 cells with (+Dox) or without (−Dox) 7 days of doxycycline induction were solubilized using increasing amounts of digitonin as indicated and analyzed by BN-PAGE and Western blotting using antibodies against mitofilin and Hsp60. The same mitochondria were also analyzed by SDS-PAGE and Western blotting using antibodies against mitofilin, Sam50, and F1α as a loading control. (C) Samples of mitochondria isolated from untreated (−Dox) or Dox-treated (+Dox) sam50kd-2 cells were solubilized in 2% digitonin buffer and separated by BN-PAGE in the first and SDS-PAGE in the second dimension. After transfer by Western blotting, the membranes were probed for mitofilin and cytochrome c as an internal control. (D) Fifty micrograms of sam50kd-2 −Dox and +Dox mitochondria described for panel A were solubilized in 1% digitonin buffer and analyzed by BN-PAGE in the first dimension and SDS-PAGE in the second dimension, followed by Western blotting using antibodies against mitofilin, Sam50, and Tom40. Individual images were overlaid to enable better comparison between complexes.

CHCHD3 exists in the same protein complex as Sam50 and mitofilin. (A) Mitochondria isolated from noninduced (−Dox) and doxycycline-induced (+Dox) tom40kd-2 cells (5 days of induction) and sam50kd-2, mflkd-2, and mtx2kd-2 cells (7 days of induction) were incubated with ³⁵S-labeled CHCHD3 for the indicated time periods and analyzed by BN-PAGE and autoradiography. Western blot analyses show the efficiency of the respective protein knockdown. Metaxin 1 levels were used to estimate the efficiency of the metaxin 2 knockdown. Decoration with Hsp60 or Tom40 antibodies served as a loading control. (B) Mitochondria isolated from HeLa cells were incubated with ³⁵S-labeled CHCHD3 for 1 h before solubilization in 1% digitonin buffer. One sample was left untreated (/), and others were treated with PBS buffer or with antibodies against mitofilin, Sam50, and Tom20. Samples were then analyzed by BN-PAGE and autoradiography. Irrelevant lanes were excised. (C) Mitochondria isolated from CHCHD3kd-2, sam50kd-2, and mflkd-2 cells after 7 days of Dox induction were analyzed by SDS-PAGE or BN-PAGE and Western blotting using antibodies against CHCHD3, mitofilin, Sam50, SDHA, and Tom40. The occasionally observed larger complex is marked with an asterisk.

Levels of Sam50 influence the amounts of respiratory complexes. Mitochondria were isolated after 7 days of knockdown induction with doxycycline (Dox) from sam50kd-2, mflkd-2, and mtx2kd-2 cells (see also A). Fifty micrograms of mitochondrial protein was solubilized in 1% digitonin buffer and analyzed by BN-PAGE and Western blotting. NDUFA9, subunit 9 of alpha subcomplex of complex I; CoxI, subunit 1 of complex IV. See the legends for and for other designations.

Assembly of respiratory complexes I and IV is affected after depletion of Sam50, mitofilin, and metaxins. (A, B, C, and D) NDUFS1 (Fe-S protein 1 of NADH dehydrogenase [ubiquinone]) and CoxVIa (cytochrome c oxidase subunit VIa) were transcribed/translated in vitro in the presence of [³⁵S]methionine/[³⁵S]cysteine. Radiolabeled proteins were imported for indicated time periods into mitochondria isolated from tom40kd-2 cells induced for 5 days with doxycycline (Dox) (A) or sam50kd-2, mflkd-2, and mtx2kd-2 cells Dox-induced for 7 days (B, C, and D). Mitochondria were solubilized in 1% digitonin buffer and analyzed by BN-PAGE and autoradiography. CIV, respiratory complex IV; CI/CIII₂/CIV_0–4, supercomplexes of respiratory complexes I, III, and IV.

Sam50-depleted mitochondria are not significantly impaired in protein import or membrane potential. (A) Mitochondria were isolated from sam50kd-2 cells grown in the absence (−Dox) or in the presence (+Dox) of doxycycline for 7 days. Mitochondria were incubated with precursors of radiolabeled ferredoxin and F1β for the indicated time periods. In the case of F1β, mitochondria were treated with protease K after import. Membrane potential (Δψ) was dissipated using 1 μM CCCP and 1 μM valinomycin. (B) Noninduced and sam50kd-2 cells induced with Dox for 7 days were seeded on a 96-well plate to the same density. Cells were stained with the Δψ-sensitive dye tetramethyl rhodamine methyl ester (TMRM). The graph represents the mean value ± SD of fluorescence signals from noninduced (−Dox) or induced (+Dox) samples normalized against the fluorescence intensity of the sample of −Dox cells with the strongest signal.

Cardiolipin levels and species are mildly affected after reduction of Sam50. Levels and distribution of cardiolipin species were measured in 5 independent samples of sam50kd-2 cells grown in the absence (−Dox) or presence (+Dox) of doxycycline for 7 days. The graph shows the mean values ± SD.

Depletion of respiratory proteins from complexes I and IV does not significantly affect mitochondrial crista morphology. (A and B) NDUFS1kd-1 (pool) and CoxVakd-2 (single clone) cells were grown for 7 days in the absence (−Dox) or presence (+Dox) of doxycycline. Cells were then analyzed by TEM (lower panels), and mitochondria were isolated in parallel and analyzed by SDS-PAGE and Western blotting. Scale bar, 1 μm. For designations, see the text and the legends for , , and .